A blycoprotein, designated BCA-225 (apparent Mr 225,000-250,000), has been identified in the cytoplasm, surface and culture medium of clone 11 T47D breast carcinoma cells by three murine monoclonal antibodies (McAs), CU18, CU26 and CU46, that have a highly restricted immunocytochemical specificity for human breast carcinoma. Since the McAs recognize different epitopes on the BCA-225 molecule, a double-determinant "sandwich" ELISA was developed with CU18 as the coating antibody and peroxidase-CU46 as the detecting antibody. A standard reference curve for the quantitation of the antigen was generated using partially purified BCA-225. Using this assay, the antigen has been found in the serum of breast cancer patients, in levels that correlate with disease status, and is either undetectable or present in very low levels in "normal" controls and patients with other malignancies. The clinical-pathological data of 1,038 patients and controls whose sera has been tested are being compiled for statistical analysis and evaluation of the clinical relevance of the assay. These data will be correlated with the immunocytochemical detection of the antigen in excised tissues. These investigations intend to continue to explore the clinical relevance of this antigen and further applications of these antibodies. Specifically, the possible significance of the increased immunocytochemical detection of BCA-225 in metastatic lesions will be studied by correlating its detection in primary and corresponding proximal and distant metastatic lesions and in serum. A longitudinal study involving prospective multiple time point serum level determinations in patients admitted for breast cancer surgery will be complemented by the immunocytochemical detection of the antigen in excised tissues. Ongoing preclinical in vivo biodistribution and imaging studies in nude mice with 111In-McAs will be continued. Other investigations will study the structural-functional relationships relating to the synthesis, intracellular distribution, glycosylation and release of BCA-225 and the epitopes recognized by the McAs, using T47D cells and factors that may influence the expression of BCA-225. Finally, the production of McAs to BCA-225, other T47D antigens and gp52 of the mouse mammary tumor virus, and their fragments, will be continued and attempts will be made to produce anti-Id and anti-anti-Id antibodies to CU18, CU26 and CU46 and explore their immunomodulating and possible immunotherapeutic potential in the BCA-225-T47D tumor model.